While cancer immunotherapy can produce dramatic responses, only a minority of patients respond to treatment. Reliable response biomarkers are needed to identify responders and conventional imaging modalities have not proved adequate. Here we provide a preclinical proof of concept for the use of granzyme B, a downstream effector of tumoral cytotoxic T cells, as a early biomarker for tumors responding to immunotherapy. We designed novel PET imaging probes for the murine and human granzyme B isoforms that specifically and quantitatively bind granzyme B. Immunotherapy-treated mice were imaged prior to therapy-induced tumor volume reduction. Imaging distinguished treated responders from non-responders with excellent predictive ability. To assess the clinical value of a granzyme B imaging paradigm, biopsy specimens from melanoma patients on checkpoint inhibitor therapy were analyzed. A marked differential in granzyme B expression was observed between treated responders and non-responders. Additionally, our human probe was able to specifically detect granzyme B expression in human samples, providing a clear candidate for clinical applcation. Overall, our results suggest granzyme B PET imaging can serve as a quantitatively useful predictive biomarker for efficacious responses to cancer immunotherapy.

Purpose: The lack of a timely and reliable measure of response to cancer immunotherapy has confounded understanding of mechanisms of resistance and subsequent therapeutic advancement. We hypothesized that PET imaging of granzyme B using a targeted peptide, GZP, could be utilized for early response assessment across many checkpoint inhibitor combinations, and that GZP uptake could be compared between therapeutic regimens and dosing schedules as an early biomarker of relative efficacy. Experimental Design: Two models, MC38 and CT26, were treated with a series of checkpoint inhibitors. GZP PET imaging was performed to assess tumoral GZP uptake, and tumor volume changes were subsequently monitored to determine response. The average GZP PET uptake and response of each treatment group were correlated to evaluate the utility of GZP PET for comparing therapeutic efficacy. Results: In both tumor models, GZP PET imaging was highly accurate for predicting response, with 93% sensitivity and 94% negative predictive value. Mean tumoral GZP signal intensity of treatment groups linearly correlated with percent response across all therapies and schedules. Moreover, GZP PET correctly predicted that sequential dose scheduling of PD-1 and CTLA-4 targeted therapies demonstrates comparative efficacy to concurrent administration. Conclusions: Granzyme B quantification is a highly sensitive and specific early measure of therapeutic efficacy for checkpoint inhibitor regimens. This work provides evidence that GZP PET imaging may be useful for rapid assessment of therapeutic efficacy in the context of clinical trials for both novel drugs as well as dosing regimens.

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Inhibitors of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway hold promise for the treatment of breast cancer, but resistance to these treatments can arise via feedback loops that increase surface expression of the receptor tyrosine kinases (RTK) epidermal growth factor receptor 1 (EGFR) and human epidermal growth factor receptor 3 (HER3), leading to persistent growth pathway signaling. We developed PET probes that provide a method of imaging this response in vivo, determining which tumors may use this escape pathway while avoiding the need for repeated biopsies. Methods: Anti-EGFR-F(ab′) 2 and anti-HER3-F(ab′) 2 were generated from monoclonal antibodies by enzymatic digestion, conjugated to DOTA, and labeled with 64 Cu. A panel of breast cancer cell lines was treated with increasing concentrations of the AKT inhibitor GDC-0068 or the PI3K inhibitor GDC-0941. Pre-and posttreatment expression of EGFR and HER3 was compared using Western blot and correlated to probe accumulation with binding studies. Nude mice xenografts of HCC-70 or MDA-MB-468 were treated with either AKT inhibitor or PI3K inhibitor and imaged with either EGFR or HER3 PET probe. Results: Changes in HER3 and EGFR PET probe accumulation correlate to RTK expression change as assessed by Western blot (R 2 of 0.85-0.98). EGFR PET probe PET/CT imaging of HCC70 tumors shows an SUV of 0.32 ± 0.03 for vehicle-, 0.50 ± 0.01 for GDC-0941-, and 0.62 ± 0.01 for GDC-0068-treated tumors, respectively (P , 0.01 for both comparisons to vehicle). HER3 PET probe PET/ CT imaging of MDAMB468 tumors shows an SUV of 0.35 ± 0.02 for vehicle-and 0.73 ± 0.05 for GDC-0068-treated tumors (P , 0.01). Conclusion: Our imaging studies, using PET probes specific to EGFR and HER3, show that changes in RTK expression indicative of resistance to PI3K and AKT inhibitors can be seen within days of therapy initiation and are of sufficient magnitude as to allow reliable clinical interpretation. Noninvasive PET monitoring of these RTK feedback loops should help to rapidly assess resistance to PI3K and AKT inhibitors and guide selection of an appropriate combinatorial therapeutic regimen on an individual patient basis.

Nivolumab has recently received approval by the Food and Drug Administration for treatment of advanced hepatocellular carcinoma (HCC) in patients previously treated with sorafenib. Nivolumabs' overall response rate of 20% (El-Khoueiry et al. in Lancet 389:2492-2502, 2017) is a step forward for these patients, but there is significant room for improvement. We describe a case of combining Y-90 radioembolization with nivolumab for treatment of angioinvasive HCC, which successfully bridged patient to partial hepatectomy. Surgical pathology showed negative margins with complete pathological response. With the introduction of immunotherapy for HCC, combining Y-90 radioembolization with immunotherapy may enhance the anti-tumoral immune response of checkpoint inhibitors.

BackgroundCancer immunotherapy research is expanding to include a more robust understanding of the mechanisms of treatment response and resistance. Identification of drivers of pro-tumor and anti-tumor immunity during treatment offers new strategies for effective alternative or combination immunotherapies. Currently, tissue or blood samples are collected and analyzed, then dichotomized based on clinical end points that may occur months or years after tissue is collected. While overall survival is ultimately the desired clinical outcome, this dichotomization fails to incorporate the nuances that may occur during an anti-tumor response. By failing to directly measure immune activation at the time of sampling, tumors may be misclassified and potentially obscure important biological information. Non-invasive techniques, such as positron emission tomography (PET), allow for global and quantitative measurements of cancer specific processes and are widely used clinically to help manage disease.MethodsWe have previously developed a novel PET agent that can non-invasively quantify granzyme B release in tumors and have demonstrated its ability to predict response to checkpoint inhibitor therapy in multiple murine models of cancer. Here, we used the quantitative measurement of granzyme B release as a direct and time-matched marker of immune cell activation in order to determine immune cell types and cytokines that correlate with effective checkpoint inhibitor therapy in both tumors and tumor-draining lymph nodes.ResultsThrough PET imaging, we were able to successfully distinguish distinct microenvironments, based on tumor type, which influenced immune cell subpopulations and cytokine release. Although each tumor was marked by functionally distinct pathways of immune cell activation and inflammation, they also shared commonalities that ultimately resulted in granzyme B release and tumor killing.ConclusionsThese results suggest that discrete tumor immune microenvironments can be identified in both responsive and non-responsive tumors and offers strategic targets for intervention to overcome checkpoint inhibitor resistance.

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Purpose: Cancer immunotherapy has markedly improved the prognosis of patients with a broad variety of malignancies. However, benefits are weighed against unique toxicities, with immune-related adverse events (irAE) that are frequent and potentially life-threatening. The diagnosis and management of these events are challenging due to heterogeneity of timing onset, multiplicity of affected organs, and lack of non-invasive monitoring techniques. We demonstrate the use of a granzyme B–targeted PET imaging agent (GZP) for irAE identification in a murine model. Experimental Design: We generated a model of immunotherapy-induced adverse events in Foxp3–DTR–GFP mice bearing MC38 tumors. GZP PET imaging was performed to evaluate organs non-invasively. We validated imaging with ex vivo analysis, correlating the establishment of these events with the presence of immune infiltrates and granzyme B upregulation in tissue. To demonstrate the clinical relevance of our findings, the presence of granzyme B was identified through immunofluorescence staining in tissue samples of patients with confirmed checkpoint inhibitor–associated adverse events. Results: GZP PET imaging revealed differential uptake in organs affected by irAEs, such as colon, spleen, and kidney, which significantly diminished after administration of the immunosuppressor dexamethasone. The presence of granzyme B and immune infiltrates were confirmed histologically and correlated with significantly higher uptake in PET imaging. The presence of granzyme B was also confirmed in samples from patients that presented with clinical irAEs. Conclusions: We demonstrate an interconnection between the establishment of irAEs and granzyme B presence and, for the first time, the visualization of those events through PET imaging.